Transfer RNA (tRNA) Fragments Are Connected to Diseases

  • Specifically Formed tRNA Fragments (tRFs) can Repress Expression by RNAi 
  • Specific tRFs are Associated with Cancer and Other Diseases
  • Chemical Modifications in tRFs Pose a Challenge for Sequencing 

Researching new, trending topics for Zone in with Zon rewards me in several ways, including learning about important subject matter that I only vaguely knew about, or had been completely unaware of. The present blog is about tRNA fragments (tRFs), which was totally new subject matter for me that I found to be very interesting and worth sharing here.

But before getting to biological formation and functions of tRFs, I want to mention what led me to this intriguing class of RNA molecules. In a nutshell, TriLink’s R&D team decided to “brainstorm” on how its expertise in chemically modified RNA might be leveraged into new product offerings beyond its current lines of modified oligo RNA and modified messenger RNA (mRNA). Since tRNAs were long known to have numerous types of chemical modifications, as detailed elsewhere, TriLink’s R&D started to think about tRFs for reasons outlined below.

Biogenesis of tRFs

Formation of tRNA is a complex process. Initially, tRNA is transcribed in the form of a precursor (pre-tRNA) containing 50-nt leader and 30-nt trailer sequences, and in some cases introns in the anticodon loop. Pre-tRNAs then undergo various types of RNA processing steps to ultimately form mature tRNAs. During tRNA maturation, the 50-nt trailer is processed by RNase P, the 30-nt trailer is removed by RNase Z, and following 30-trailer removal, the 30-nt end of all human tRNAs is modified by enzymatic addition of the universal CCA triplet, as depicted here.

Pre-tRNA (left) and mature tRNA (right); adapted from Anderson & Ivanov FEBS Lett (2014)

Also depicted here are specific types of enzymatic cleavage reactions of mature tRNA by ribonucleases Dicer and angiogenin (ANG) that lead to formation of 5’-tRFs and 3’-CCA tRFs, as well as 5’-halves and 3’-halves. These tRFs derived from mature tRNAs, as well as tRFs from pre-tRNAs that will not be discussed here, have now been extensively characterized by high-throughput short RNA sequencing methods. Among new advances in this sequencing methodology, TriLink’s recent PLOS One publication of its innovative CleanTag™ sample prep procedure has already been viewed an impressive ~6,000 times since appearing online only ~14 months ago as of this writing.

Mature tRNA (adapted from Anderson & Ivanov)

It should be noted that tRFs are not restricted to humans but have been shown to exist in multiple organisms. Two online tools are available for those wishing to learn more about tRFs: the framework for the interactive exploration of mitochondrial and nuclear tRNA fragments (MINTbase) and the relational database of Transfer RNA related Fragments(tRFdb). MINTbase also provides a scheme for the naming of tRFs called tRF-license plates that is genome independent. A recent publication by Kim et al. is a good lead reference for various functions of tRFs, some of which include the following.

Possible Roles of tRFs in Human Diseases

In a review of this subject, Anderson & Ivanov emphasize that, while production of tRFs have been observed in several types of human diseases, it remains to be determined whether these tRFs contribute to disease pathogenesis. Landmark findings regarding functions of tRFs were published by a team, including Andrew Fire—2006 Nobel Laureate for  RNA interference (RNAi)—titled Human tRNA-derived small RNAs in the global regulation of RNA silencing that provided compelling evidence demonstrating that human tRFs can enter RNAi pathways. These findings by Fire & coworkers are now recognized as a previously unknown nexus of RNAi translational repression pathways involving tRFs and microRNAs (miRNAs) depicted here.

Schematic representation of the biogenesis of miRNAs and tRFs associated with Argonaute (AGO) proteins. Taken from Shigematsu & Kirino Gene Regul Syst Bio (2015)

tRFs and Cancer: In 2009, Lee et al. reported that a specific tRF, designated as tRF-1001, is highly expressed in a wide range of cancer cell lines but much less in tissues, and its expression in cell lines was tightly correlated with cell proliferation. Furthermore, siRNA-mediated knockdown of tRF-1001 impaired cell proliferation. Since that discovery, various research groups have similarly found specific tRFs associated with different types of cancer, as recently detailed by Croce & coworkers, who concluded the following:

“We found that tRNA-derived small RNAs (tsRNAs) [i.e. tRFs in this blog] are dysregulated in many cancers and that their expression is modulated during cancer development and staging. Indeed, activation of oncogenes and inactivation of tumor suppressors lead to a dysregulation of specific tRFs, and tRFs-knock out cells display a specific change in gene-expression profile. Thus, tRFs could be key effectors in cancer-related pathways. These results indicate active crosstalk between tRFs and oncogenes and suggest that tRFs could be useful [bio]markers for diagnosis or targets for therapy. Additionally, [overexpression of two specific tRFs] affect cell growth in lung cancer cell lines, further confirming the involvement of tRFs in cancer pathogenesis.”

Biomarkers in blood, which I’ve blogged about previously, are a “hot topic” in disease diagnostics because they offer a more general, less invasive and safer means of patient sample access compared to traditional tumor biopsies.

tRFs and Pathological Stress Injuries: Stress-related cellular damage is central to disease pathogenesis that can be induced by hypoxia, nutrient deprivation, oxidative conditions and metabolic imbalance. Dhahbi et al. sequenced short RNAs from mouse serum and identified abundant 5′-halves derived from a small subset of tRNAs, implying that these tRFs are produced by tRNA type-specific biogenesis. A survey of somatic tissues revealed that these tRFs are concentrated within blood cells and hematopoietic tissues, with very little in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of these 5′ tRNA halves change markedly with age, either up or down, and these changes were prevented by calorie restriction.

Taken from Mishima et al. J Am Soc Nephrol (2014)

In a study by Mishima et al., it was shown in vivo that oxidative stress leads to conformational changes in tRNA that thus allows ANG-mediated productin of tRFs. This stress-induced conformational change allows 1-methyladenosine nucleoside (m1A), a modification important for stabilizing the L-shaped structure of tRNA, to be recognized by an m1A-specific antibody, as depicted here. This antibody was used to show that renal injury and cisplatin-mediated nephrotoxicity (which both induce tissue damage via oxidative stress) generate tRFs. Similar results were obtained using m1A-based immunohistochemistry to directly visualize damaged areas of kidneys, brain and liver. Mishima et al. further demonstrated that these tRFS avoid degradation in the blood because they are associated with circulating exosomes, which are extracellular vesicles packed with proteins and nucleic acids.

tRFS and Neurodegenerative Diseases: As detailed in the above mentioned review by Anderson & Ivanov, ANG mutants possessing reduced ribonuclease activity were reported in 2006 to be implicated in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS; aka Lou Gehrig disease), which is a fatal neurodegenerative disease that I have blogged about. In 2012, a subset of ALS-associated ANG mutants was also found in Parkinson’s Disease (PD) patients. Recombinant ANG is neuroprotective for cultured motor neurons, and administration of ANG to a standard mouse model for ALS significantly promotes both life-span and motor function.

Concluding Comments on Analysis of tRFs

Although I started this blog by refering to the fact that mature tRNAs are extensively modified by a wide variety of nucleobase and ribose chemical modifications, these modifcations were not further mentioned. That is because sample prep for short RNA sequencing uses reverse transcription to form cDNA that is then PCR amplified before sequencing, and it is widely acknowledged (e.g. Cozen et al.) that certain chemical modifications in RNA can interfere with reverse transcription. Thus, aside from reported use of demethylases to first remove interfering methyl groups from m1A, N1-methylguanosine, N3-methylcytosine, and N2,N2-dimethylguanosine, sequenced tRFs exclude many tRFs having chemical modifications that prevent reverse transcription.

Recognizing the need for alternative methods of determining structures of chemically modified tRFS, Limbach & Paulines have recently proposed the possibility of developing mass spectrometric (aka mass spec) approaches in a publication provocatively titled Going global: the new era of mapping modifications in RNA. I think this is a great idea, and hope that the mass spec community will soon address this challenge.

As usual, your comments are welcomed.


After writing this blog, Eng et al., who investigated the mosquito Aedes aegypti—the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses—reported the following:

Aedes aegypti mosquito. Taken from

“[A]single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology.”

Click here to read my past blogs about Zika virus.

Jerry’s Favs from the Recent OTS Meeting

  • 12th Annual OTS Meeting in Montreal Très Excitant!
  • RNAi Approach to Treating Preeclampsia Spurred by Researchers’ Personal Experiences
  • Ionis Video for Spinal Muscular Atrophy Amazes the Audience
  • 30 years later, PS-Modified Oligonucleotides Continue to be Enabling!

City line of Montreal. Taken from

The 12th Annual Meeting of the Oligonucleotide Therapeutics Society (OTS) held on September 25-28, 2016 in French-speaking Montreal, Quebec, Canada brought together hundreds of enthusiastic investigators from around the world. All attendees share a common interest in oligo-based therapeutics, and we gratefully say merci beaucoup to the organizers of this very well run event.

Commenting here on my “favs” is limited by space, and highly subjective—by intent—focusing on only several impressions that struck me as worth sharing. Readers interested in perusing all of the lecture titles and speaker biosketches can do so at this link, which also lists corporate sponsors—including TriLink—and connects with the regularly updated OTS website.

Jerry’s balcony view of OTS 2016 presentations at the Centre Mont-Royal venue

Jerry’s balcony view of OTS 2016 presentations at the Centre Mont-Royal venue

Before getting to my selected topics, I wish to congratulate the OTS Board of Directors and Scientific Advisory Council for their ongoing valuable contributions to this society, and continuing efforts to include participation by the new generation of young investigators, who I’m sure will collectively make exciting advances in the field of oligonucleotide therapeutics. It was pleasure for me to me meet some of these “next gen” scientists, and learn about their current work, which is quite sophisticated by comparison to what I and others did in the early—dare I say primitive—era of antisense oligonucleotide drug discovery.

Toward Treating Preeclampsia: Personalized Drug Developers’ Stories

Preeclampsia (PE) is a disorder that occurs during pregnancy, affects both the mother and the fetus, and is characterized by elevated blood pressure, swelling and protein in the urine. According to a Preeclampsia Foundation fact sheet, every minute somewhere in the world a woman dies in pregnancy or childbirth, which amounts to more than 500,000 deaths each year. In addition, PE causes ~15% of premature births in industrialized countries and is the number one reason doctors decide to deliver a baby early.

Dr. Melissa J. Moore is a professor in the RNA Therapeutics Institute and the Department of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School. Taken from

Dr. Melissa J. Moore is a professor in the RNA Therapeutics Institute and the Department of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School. Taken from

In her OTS lecture on clinical development of an RNA interference (RNAi) approach to treat PE, Dr. Melissa J. Moore (pictured below) introduced an attention-grabbing personal element when she revealed her bout with PE. Coincidentally, she discovered that her then attending physician, Dr. S. Ananth Karumanchi, had begun his quest for identifying PE-causality at the molecular level prompted by his daughter’s dangerously premature birth due to PE. This turned out to be a truncated kinase receptor abbreviated sFlt1, which Moore described as Karumanchi’s break-through discovery for possible development of PE therapeutics.

The condensed version of these two remarkably interwoven, PE-related personal stories—that you can hear first-hand from Moore on YouTube—was an agreement between Moore and Karumanchi to collaborate on discovering whether double-stranded short-interfering RNA (siRNA) targeting sFlt1 could be useful as an RNAi-based PE therapeutic agent.

Melissa Moore’s ultrasound sonogram. Taken from her YouTube video

Melissa Moore’s ultrasound sonogram. Taken from her YouTube video

Moore went on to gratefully acknowledge her colleague at the RNA Therapeutics Institute, Prof. Anastasia Khvorova, for setting up “by hook or by crook” the first non-profit academic facility for large-scale production of siRNA suitable for clinical development. This led to designing and producing a nuclease-resistant siRNA comprised of 2’-OMe/2’-F ribonuceosides and phosphorothioates at the ends, with an attached hydrophobic cholesterol moiety for improved delivery.

Even more impressive—at least to me—was Moore’s ability to access pregnant baboons in a non-human primate model of PE. This is inherently difficult due to issues involving non-human primates for any research, and is technically much more challenging compared to, for example, infectious disease models. Dr. Moore showed lots of compelling results from her model studies, and concluded her talk by saying that clinical studies were planned.

In closing this section, it’s worth noting that Moderna—a leading mRNA therapeutics company—has recently announced its appointment of Dr. Moore as Chief Scientific Officer of Moderna’s mRNA Research Platform.

Ionis Pharmaceuticals Drug Video for Spinal Muscular Atrophy Amazes the Audience

Dr. Stanley T. Crooke. Taken from

Dr. Stanley T. Crooke. Taken from

This year’s OTS Lifetime Achievement Award address by Dr. Stanley T. Crooke, founder and CEO of Ionis Phamaceuticals (formerly Isis Pharmaceuticals), was presented to an auditorium packed with attendees who, like me, greatly admire Crooke’s many scientific and commercial contributions to the field over the past decades. These currently include more than 450 (!) scientific publications and 38 drugs in the pipeline, with 3 finishing Phase III—very impressive and promising indeed!

Ionis’ impact on providing efficacious oligonucleotide-based therapies to patients was conveyed most powerfully—in my opinion—by a video about Cameron, an infant SMA patient who was born with Spinal Muscular Atrophy (SMA). According to an NIH fact sheet, SMA is a genetic disease that causes weakness and wasting of the voluntary muscles in the arms and legs of infants and children. These disorders are linked to an abnormal or missing gene known as the survival motor neuron gene 1 (SMN1), without which motor neurons in the spinal cord degenerate and die.

As detailed elsewhere by Chiriboga et al., Nusinersen (aka ISIS-SMNRx or ISIS 396443) is a 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) designed to alter splicing of SMN2 mRNA and increase the amount of functional SMN protein produced, thus compensating for the genetic defect in the SMN1 gene. The cartoon shown below depicts how this ASO targets an hnRNP-A1/A2–dependent splicing silencer in intron 7 of the SMN pre-mRNA. Nusinersen displaces hnRNP proteins from this silencer site on the SMN2 pre-mRNA, facilitating accurate splicing of SMN2 transcripts (e.g., increasing the synthesis of transcripts containing exon 7) and resulting in increased production of full-length SMN protein.

Taken from after Chiriboga et al.

Taken from after Chiriboga et al.

Ionis and its commercial partner Biogen announced in August 2016 that Nusinersen met the primary endpoint pre-specified for the interim analysis of ENDEAR, the Phase 3 trial evaluating Nusinersen in infantile-onset (Type 1) SMA. The analysis found that infants receiving Nusinersen experienced a statistically significant improvement in the achievement of motor milestones compared to those who did not receive treatment. In September, Biogen announced that it applied for Priority Review by the FDA that, if granted, would shorten the review period of Nusinersen following the Agency’s acceptance of the NDA filing by Ionis and Biogen.

Cameron at 2+ years enjoying the moment with his mom. Taken from YouTube

Cameron at 2+ years enjoying the moment with his mom. Taken from YouTube

The amazing effect of Nusinersen warranting this Priority Review is best appreciated—in my opinion—by watching a YouTube video showing the progress of Cameron during his treatment. When viewing this video, keep in mind that Cameron’s Type I SMA is typically evident at birth or within the first few months, and that symptoms include floppy limbs and trunk, feeble movements of the arms and legs, swallowing and feeding difficulties, and impaired breathing. Sadly, the prognosis is poor for babies with SMA Type I. Most die within the first two years. But not so for Cameron, shown below at 2+ years, thanks to Nusinersen!

Phosphorothioate-Modified Oligonucleotides Continue to be Enabling—30 Years On!


Left: Prof. Fritz Eckstein. Taken from Right: Prof. Wojciech J. Stec. Taken from

That nuclease-resistant phosphorothioate (PS)-modified internucleotide linkages have had an enabling influence on all manner of oligo-based drug development is evident from the first-ever OTS Lifetime Achievement Award in 2015 being given to Prof. Fritz Eckstein for his pioneering and life-long work on PS linkages in DNA and RNA. Following Fritz’s lead, and aided by the availability of ABI’s DNA synthesizer, Prof. Wojciech J. Stec and yours truly published the first completely automated method for synthesis of fully or partially PS-modified DNA oligos in 1984. This opened the door for early antisense experiments with PS-modified oligos, which have continued now for 30 years!

Perhaps surprisingly, the ever evolving field of oligonucleotide therapeutics continues to rely on PS-modifications in a myriad of mechanistically distinct strategies, such as “classic” antisense to mRNA, siRNA, anti-miRNAs, modulators of RNA splicing, etc. In this regard, Dr. Crooke’s concluding remarks on his goal of deciphering the “code” for protein binding to chemically modified ASO, led me to muse about the negatively charged P-Smoiety in such binding of PS-containing ASO.

Taken from

Taken from

My initial thought was that P-S likely enhances binding to positively charged amino acid moieties in proteins, due to greater polarizability vs. P-O, and that Sp or Rp P-Sstereochemistry may likely influence binding. Moreover, this spatial aspect of a binding “code” can now be studied using stereospecific synthesis of PS-ASO by either Stec’s OTP method or Wada’s oxazaphospholidine method. Time will tell—stay tuned!
As usual, your comments here are welcomed.

Curiously Circular RNA

  • Circular RNA (circRNA) Formation Serendipitously Discovered in 1991  
  • Next-Generation Sequencing Reveals circRNA to be Ubiquitous
  • circRNA can Function as MicroRNA ‘Sponges’ to Regulate Gene Expression

There’s something seductively simple—and curious—about circles, which are unique in having no beginning or end, unlike most other things. On a less philosophical plane, thinking about circles conjures up incongruent memories of delicious doughnuts and geometric definitions from my youthful days going to the neighborhood bakery and diligently taking notes in my high school geometry class, respectively.


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Profiling Pseudouridine

  • Two New Methods for Sequencing Pseudouridine Leverage Old Chemistry
  • New Methods Reveal ‘Rewiring’ of Genetic Code by Post-Transcriptional Pseudouridination
  • Exciting Future for New Analytical Methods for Modified mRNA
  • Be Sure to Read the Very End of the Blog for a Special Offer!

At the risk of seeming enamored with pseudouridine, which I previously proclaimed—with justifications—to be The 2014 Modified Nucleobase of the Year, recent reports about this fascinating base lead me now to feature it here once again. In that past post, it was pointed out that uridine, which is incorporated into all RNA during transcription of genomic DNA, differs from pseudouridine—historically abbreviated by the Greek symbol Ψ –by how one nitrogen (shown in red below) switches place with a carbon for bonding to the ribose ring. It was also noted that this switch has been long known to be carried out after transcription (aka post-transcriptionally) by an enzyme called—appropriately—pseudouridine synthase, the exact mechanistic details for which remain controversial. This post-transcriptional process that converts U to Ψ at specific positions in RNA is called pseudouridination.
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Nanopore Sequencing: 20 Years On 

  • Once Only a Dream, Nanopore Sequencing is Now Reality
  • Oxford Nanopore Technologies MinION Sequencer Apps Spotlighted  
  • Fully Automated Sample Prep and Higher Throughput Coming Soon
Dream Messenger oil painting by Leszek Andrzej Kostuj with nanopore-like eyes. Taken from

Dream Messenger oil painting by Leszek Andrzej Kostuj with nanopore-like eyes. Taken from

I don’t really know who first dreamt the exciting idea of moving DNA through a nanometer-sized pore to read sequence as electronic blip-like signals, but lots of folks are glad that someone did because this seemingly impossible dream is now reality.

My initial close encounter with this almost alien idea—pun intended—happened in 1998 when I first read about it in a patent by George Church (my recent blog-pick as The Most Interesting Scientist in the World), David Deamer, Daniel Branton, and others jointly assigned to Harvard and the University of California. This patent was filed 20 years ago on March 17, 1995 so St. Patrick’s Day should share with what I hereby suggest as annual Nanopore Sequencing Day.
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Pseudouridine: 2014 Modified Nucleobase of the Year

  • A Minor Modified Nucleobase Playing a Major Role in RNA Therapeutics
  • Nucleobase is “Acrobatically” Formed in RNA by an Enzyme
  • Its Original Identification is Linked to the Atomic Bomb! 

The Molecule of the Year was started in 1989 by Science magazine and in 1996 was changed to ‘Breakthrough of the Year’. After a brief hiatus, the Molecule of the Year designation was revived in 2002 by the International Society for Molecular and Cell Biology and Biotechnology Protocols and Research. If you peruse the list of these lauded molecules, you’ll find that DNA has received this coveted award twice: “PCR and DNA polymerase” (1989) and “DNA repair enzyme” (1994).

While the Molecule of the Year is fascinating and well deserved, I think we should show some love for individual nucleobases given that this blog focuses on “what’s trending in nucleic acid research”—and since TriLink scientists are “The Modified Nucleic Acid Experts”. So, I thought it would be apropos (and fun) to start an annual post on Modified Nucleobase of the Year.

After mulling over which modified nucleobase merits this accolade for 2014, I decided on pseudouridine (aka 5-ribosyluracil)—an unusual isomer of uridine—for several reasons that are given below, followed by some history of its discovery that I found to be quite interesting.


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Reflections on Advances in Medicinal Oligonucleotides

  • Oligos Are Not “Magic Bullets”
  • Oligos Have, Nevertheless, Enabled New Drug Paradigms
  • Oligos Continue to Attract Significant Corporate Investments


The 10th Annual Meeting of the Oligonucelotide Therapeutics Society (OTS) is in full swing today in San Diego, CA where it began on Oct 12 and concludes on Oct 15. Having worked on antisense oligos since the early days (~30 years ago) participating in this meeting led me to several thoughts that I wish to share with you in this post.

First of all, contrary to many sceptics in those early days, the concept of using synthetic oligonucleotides as an entirely new class of medicinal agents has not only survived but also greatly expanded in terms of the biological target/mechanism of action and types of oligo constructs used—each with a seemingly endless array of chemical modifications to evaluate. In approximate chronological order of discovery, these targets and the types of oligos they have now come to include are listed below coincidentally, most of these are represented in the 2014 OTS agenda.

transcription factors
splice junctions
RNA interference
Oligo Type
dsDNA decoys






Secondly, while it has been possible for oligo chemists to design and synthesize a plethora of modified oligos to achieve optimized nuclease stability, binding to target, etc., efficient delivery has remained the single most challenging problem to deal with. In talks on medicinal oligos, this situation is oftentimes eluded to as something to the effect of “there are only three remaining problems to solve: delivery, delivery, and delivery.”

Lastly, contrary to early hopes of being Dr. Ehrlich’s “magic bullets” (see caption below), oligos didn’t quite prove to be the new paradigm for a speedy concept-to-clinic solution. As all of us in the oligo world know, oligo-based therapeutics have encountered long and costly R&D timelines and clinical development paths typical of all other classes of therapeutic compounds.

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The Elusive Commercial Pursuit of RNAi Drugs

  • No Synthetic siRNA Drug Approval Some 13 Years “A.T.” (After Tuschl)
  • Alynlam Buys Sirna RNAi Assets from Merck and Forges Alliance with Genzyme
  • Novartis Cuts Back on In-House RNAi Efforts
  • Will RNAi for Therapeutics or for AgBio Pay-Off First?

Discovery, development and successful clinical investigations leading to new drugs are long and costly endeavors. In a detailed overview provided by The Pharmaceutical Research and Manufacturers of America (PhRMA), this process generally takes 10-15 years—at best. None of this work is cheap, and a relatively recent article in Forbes is critical of the drug industry “tossing around the $1 billion number for years.” The article digs into Big Pharma data to show that actual costs can reach $15 billion when failed drugs and all R&D are taken into account.

Thomas Tuschl (taken from The Rockefeller University via Bing Images).

Thomas Tuschl (taken from The Rockefeller University via Bing Images).

Having said this, it’s no surprise that seeking the first oligonucleotide drug based on RNA interference (RNAi) is still elusive today, some 13 years after Thomas Tuschl and collaborators at the Max-Planck-Institute for Biophysical Chemistry in Göttingen, Germany first reported use of synthetic 21-nucleotide RNA duplexes for RNAi. Their landmark publication in Nature in 2001, which has been cited over 9,000 times, demonstrated that these short-interfering RNA (siRNA) duplexes specifically suppressed expression of endogenous and heterologous genes in different mammalian cell lines. They presciently concluded that “21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.”

While siRNA did in fact become an amazingly powerful “new tool” very quickly, couching potential therapeutic utility of siRNA as an outcome that “may eventually” occur has indeed proven apropos.

Thousands of publications have led to elucidation of molecular pathways for RNAi offering various possible mechanisms of action for other types of RNAi agents. That—and delivery approaches for RNAi clinical candidates being investigated—can be read about in an excellent review by Rossi and others. The focus of this post is the elusive commercial pursuit of an RNAi drug.

Four Phases of the Business of RNAi Therapeutics

The elusive nature of RNAi therapeutics is not for lack of trying or underinvestment.  According to Dirk Haussecker, ‘the business of RNAi therapeutics’ has gone through four phases, which he explores in his excellent account entitled The Business of RNAi Therapeutics in 2012. His views in that article are paraphrased as follows:

It all began with the discovery phase (2002–05), which was defined by the early adopters of RNAi as a therapeutic modality. These were small, risk-taking biotechnology companies such as Ribozyme Pharmaceuticals (aka Sirna Therapeutics), Atugen (aka Silence Therapeutics) and Protiva (aka Tekmira). As much as they may have believed in the potential of RNAi therapeutics, their strategic reorientation was also a gamble on a technology with considerable technical uncertainties in hopes of turning around declining business fortunes by leveraging their nucleic acid therapeutics know-how to become leaders in a potentially disruptive technology. This phase also saw the founding of Alnylam Pharmaceuticals—by Thomas Tuschl, Phillip Sharp (1993 Nobel Prize), and others—based on the idea of cornering the IP on the molecules that mediate RNAi so that it may finance its own drug development by collecting a toll from all those engaged in RNAi therapeutics.

Left-to-right: Craig Mello, Andrew Fire, and Alfred Nobel (taken from via Bing Images).

Left-to-right: Craig Mello, Andrew Fire, and Alfred Nobel (taken from via Bing Images).

Big Pharma initially saw the value of RNAi largely as a research tool only, but this quickly changed. The defining feature of this second phase—the boom phase (2005–08)—was the impending patent cliff and the hope that the technology would mature in time to soften its financial impact. A bidding war, largely for access to potentially gate-keeping RNAi IP, erupted. Most notably, Merck acquired Sirna Therapeutics for $1.1 billion, while a Roche and Alnylam alliance provided a limited platform license from Alnylam for $331 million in upfront payments and equity investment. This boom phase was also fueled by the award of a Nobel Prize to Andrew Fire and Craig Mello for their seminal discovery of double-stranded RNA (dsRNA) as the trigger of RNAi.

This period of high expectations and blockbuster deals was followed by a backlash phase (2008–2011), or buyer’s remorse, in part due to absence of adequate delivery technologies and concerns for specificity and innate immune stimulation as safety issues. Suffering from RNAi-specific scientific and credibility issues, and with first drug approvals still years away, RNAi therapeutics was among the first to feel the cost-cutting axe. The exit of Roche from in-house RNAi therapeutics development sent shockwaves through the industry. Having invested heavily in the technology only 2–3 years ago, and being considered an innovation bellwether within Big Pharma, Roche’s decision in late 2010 found a number of imitators among Big Pharma and can be credited (or blamed, depending on your perspective), for intrepid investment in RNAi therapeutics ever since.

The backlash, incidentally, also had cleansing effects, many of which form the basis for the 4th and final phase, recovery (2011–present). This shift is most evident in the evolution of the RNAi therapeutics clinical pipeline that has become more and more populated with candidates based on sound scientific rationales, especially in terms of delivery approaches and anti-immunostimulatory strategies. For the recovery, however, to firmly take root and for the long-term health of the industry, it is important for the current clinical dataflow to bring back investors.

Current Status of RNAi Therapeutics

Dirk Haussecker’s The RNAi Therapeutics Blog richly chronicles the aforementioned and many more events dating back to 2007 and continuing through today. Particularly worth visiting is the Google-based World of RNAi Therapeutics map that shows current companies and—more importantly—the various RNAi agents under investigation. The screen shot below  exemplifies the kind of information that is displayed when you click on any company on the map—Alnylam in this case. Very convenient indeed!

Screen shot of World of RNAi Therapeutics exemplified with selection of Alnylam from the updated list of companies in the panel on the left (taken from The RNAi Therapeutics Blog).

Screen shot of World of RNAi Therapeutics exemplified with selection of Alnylam from the updated list of companies in the panel on the left (taken from The RNAi Therapeutics Blog).

It’s worth mentioning that is a web-based resource that provides patients, their family members, health care professionals, researchers, and the public with easy access to information on publicly and privately supported clinical studies on a wide range of diseases and conditions. The website is maintained by the National Library of Medicine at the National Institutes of Health. Information on is provided and updated by the sponsor or principal investigator of the clinical study. Studies are generally submitted to the website (that is, registered) when they begin, and the information on the site is updated throughout the study.

My February 2014 search of “siRNA” as a keyword at found 31 studies listed. These are initially shown as a simplified list of the clinical study name and whether each study is completed, actively recruiting, active but not recruiting, terminated, etc. The list can be easily sorted by condition (i.e. disease type), sponsor/collaborators, and other parameters. Another useful feature is viewing found studies based on geographic location, as shown below. Click on any region or sub-region (i.e., state) to view information regarding studies in that area.

Global map of siRNA clinical studies taken from

Global map of siRNA clinical studies taken from

Map of “siRNA” clinical studies in the USA taken from

Map of “siRNA” clinical studies in the USA taken from

Alnylam Ascending

The big news for 2014 in ‘the Business of RNAi’—to borrow Dirk Haussecker’s expression—will most likely be centered around two deals involving Alnylam that were announced in January. The first announcement was that Alnylam will acquire “investigational RNAi therapeutic assets” from Merck for “future advancement through Alnylam’s commitment to RNAi Therapeutics.” The acquisition of Merck’s wholly owned subsidiary, Sirna Therapeutics, provides IP and RNAi assets including pre-clinical therapeutic candidates, chemistry, siRNA-conjugate and other delivery technologies.

Under the agreement, in exchange for acquiring the stock of Sirna Therapeutics, Alnylam will pay Merck an upfront payment of $175 million in cash and equity—$25 million cash and $150 million in Alnylam common stock. In addition, Merck is eligible to receive up to $105 million in developmental and sales milestone payments per product, as well as single-digit royalties, associated with the progress of certain pre-clinical candidates discovered by Merck. Merck is also eligible to receive up to $10 million in milestone payments and single-digit royalties on Alnylam products covered by Sirna Therapeutics’ patent estate.

Merck’s decision was quoted to be “consistent with [Merck’s] strategy to reduce emphasis on platform technologies and prioritize [Merck’s] R&D efforts to focus on product candidates capable of providing unambiguous promotable advantages to patients and payers.”

Alnylam expects to have six to seven genetic medicine product candidates in clinical development—including at least two programs in Phase 3 and five to six programs with human proof of concept—by the end of 2015 and referred to as “Alnylam 5×15” programs, details for which can be accessed here and in presentations.

The second announcement, which came one day after announcing the acquisition of Sirna from Merck—was that Alnylam and Genzyme would form a “transformational alliance” for RNAi therapeutics as genetic medicines. This new collaboration is “expected to accelerate and expand global product value for the RNAi therapeutic genetic medicine pipeline, including ‘Alnylam 5×15’ programs.”

Alnylam will retain product rights in North America and Western Europe, while Genzyme will obtain the right to access Alnylam’s current “5×15” and future genetic medicines pipeline in the rest of the world (ROW), including global product rights for certain programs. In addition, Genzyme becomes a major Alnylam shareholder through an upfront purchase of $700 million of newly issued stock, representing an approximately 12% ownership position. This alliance significantly bolsters Alnylam’s balance sheet to over $1 billion in cash that was said “to increase [Alnylam’s] investment in new RNAi therapeutic programs, while securing a cash runway that [Alnylam] believes will allow [it] to develop and launch multiple products as breakthrough medicines.”

In addition to the upfront equity purchase, Alnylam will receive R&D funding, starting on January 1, 2015, for programs where Genzyme has elected to opt-in for development and commercialization. In addition, Alnylam is eligible to receive milestones totaling up to $75 million per product for regional and co-developed/co-promoted programs. In the case of global Genzyme programs, Alnylam is eligible to receive up to $200 million in milestones per product. Finally, Alnylam is also eligible to receive tiered double-digit royalties up to 20% on net sales on all products commercialized by Genzyme in its territories. In the case of Genzyme’s co-developed/co-promoted products in the Alnylam territory, the parties will share profits equally and Alnylam will book net sales revenues.

Those interested in a “deep dive” into Alnylam’s impressive array of other strategic alliances can find lead information here.

First RNAi Drug Approval on the Horizon

Hopefully, the ‘Business of RNAi’ is entering its 5th phase: drug approval for sale, which would—finally—provide long-awaited demonstration of clinical utility and commercial payback toward huge investments to date.

In this regard, Alnylam has recently begun recruiting patients for a pivotal Phase III clinical trial that could lead to the first RNAi drug approval in the near future. This comes shortly after Alnylam’s November 2013 detailed press release announcing positive data from a Phase II clinical trial of patisiran (ALN-TTR02) for the treatment of transthyretin-mediated amyloidosis (ATTR), presented at the International Symposium on Familial Amyloidotic Polyneuropathy. The 24-slide deck of this Symposium presentation can be downloaded as a pdf by clicking here. Results showed that multiple doses of a Tekmira Phamaceuticals lipid nanoparticle formulation of ALN-TTR02 led to robust and statistically significant knockdown of serum TTR protein levels of up to 96%, with mean levels of TTR knockdown exceeding 85%. Knockdown of TTR, the disease-causing protein in ATTR, was found to be rapid, dose dependent, and durable, and similar activity was observed toward both wild-type and mutant protein. In addition, ALN-TTR02 was found to be generally safe and well tolerated in this study.

Details for the Phase III multicenter, multinational, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of ALN-TTR02 can be read here at Among the details are the following facts, including the targeted completion date. While the trials look promising so far, January 2017 is several years away, and it’s wise to “never count your chickens before they hatch.”

Estimated Enrollment: 200
Study Start Date: November 2013
Estimated Study Completion Date: May 2017
Estimated Primary Completion Date: January 2017 (Final data collection date for primary outcome measure)

Novartis Cuts Back Its In-House RNAi R&D

While investment in RNAi at Alnylam is ascending, the situation at Novartis is descending, based on an article in GenomeWeb in April of 2014 stating that Novartis will be cutting back its 26 person effort. The article adds that, according to a Novartis spokesperson, the decision was driven by “ongoing challenges with formulation and delivery and the reality that the current range of medically relevant targets where siRNA may be used is quite narrow.”

Despite its decision to dial down its RNAi programs, Novartis still holds onto the rights to use Alnylam’s technology against the 31 targets covered under their one-time partnership, the company spokesperson said.

And as work continues on those targets, albeit by a downsized research team, Novartis will also considering partnering opportunities in the space, the spokesperson added.

With the seemingly never ending challenges of formulation and delivery, perhaps RNAi will pay-off first in the agricultural biotechnology (aka AgBio) space, as briefly discussed in the next section.

Pros and Cons of RNAi for AgBio

RNAi can be achieved using genetically encoded sequences rather than using chemically synthesized siRNA duplexes or other types of synthetic oligonucleotides. Agricultural biotechnology has already taken advantage of such genetically engineered constructs in producing stable and heritable RNAi phenotype in plant stocks. Analogous procedures can be applied to other organisms—including humans, such as in antiviral stratagems against HIV-1.

Andrew Pollack recently reported in the New York Times that agricultural biotechnology companies are investigating RNAi as a possible approach to kill crop-damaging insects and pathogens by disabling their genes. By zeroing in on a genetic sequence unique to one species, the technique has the potential to kill a pest without harming beneficial insects. That would be a big advance over chemical pesticides.

Subba Reddy Palli, an entomologist at the University of Kentucky who is researching the technology, is quoted as saying “if you use a neuro-poison, it kills everything, but this one is very target-specific.”

Some specialists, however, fear that releasing gene-silencing agents into fields could harm beneficial insects, especially among organisms that have a common genetic makeup, and possibly even endanger human health. Pollack adds that this controversy echoes the larger debate over genetically modified crops, which has been raging for years. The Environmental Protection Agency (EPA), which regulates pesticides, is meeting with scientific advisers to discuss the potential risks of RNA interference.

RNAi May Be a Bee’s Best Friend

Monsanto is exploring the use of RNAi to kill a mite that may play a role in bee die-offs.  Photo: Monsanto (taken from

Monsanto is exploring the use of RNAi to kill a mite that may play a role in bee die-offs. Photo: Monsanto (taken from

In addition to use in AgBio, RNAi may prove useful in reviving bee populations. RNAi is of interest to beekeepers because one possible use, under development by Monsanto, is to kill a mite that is believed to be at least partly responsible for the mass die-offs of honeybees in recent years.

In opposition to this, the National Honey Bee Advisory Board is quoted as saying “to attempt to use this technology at this current stage of understanding would be more naïve than our use of DDT in the 1950s.”

Pollack reports that some bee specialists told the EPA that they would welcome attempts to use RNAi to save honeybees, and groups representing corn, soybean and cotton farmers also support the technology: “commercial RNAi technology brings U.S. agriculture into an entirely new generation of tools holding great promise,” the National Corn Growers Association said.

Corn Growers Need a New Tool

For a decade, corn growners have been combating the rootworm, one of the costliest of agricultural pests, by planting so-called BT crops, which are genetically engineered to produce a toxin that kills the insects when they eat the crop. Or at least the toxin is supposed to kill them. Rootworms are now evolving resistance to at least one BT toxin.

Given that rootworm larvae can destroy significant percentages of corn if left untreated, a robust alternative is crucial to protecting future corn crops. Current estimates in the US indicate as much as 40% of corn acreage is infested with corn rootworms and the area is expected to grow over the next 20 years. RNAi is now is being studied as a possible alternative to BT toxins, and Monsanto has applied for regulatory approval of corn that is genetically engineered to use RNAi to kill the western corn rootworm.

Corn rootworm damage. Photo: IPM Images (taken from via Bing Images).

Corn rootworm damage. Photo: IPM Images (taken from via Bing Images).

Personally, I’m not completely averse to RNAi for AgBio, especially in view of the need to adequately feed the world’s growing population. Careful regulatory scrutiny, even if it results in slow moving progress, seems wise in order to avoid unintended consequences that could be very problematic.

As usual, your comments are welcomed.